Proteasome-mediated degradation of collagen III by cortisol in amnion fibroblasts

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   Figure 1

   Changes of COL3A1 protein abundance in human amnion tissue. (A) Protein abundance of COL3A1 in human amnion tissue was decreased following spontaneous rupture of membranes at term parturition (labor) (n = 7) in comparison with elective c section without labor at term (non-labor) (n = 9). (B) Cortisol (1 μM, 24 h) reduced COL3A1 protein abundance in cultured human amnion tissue explants n = 3. The left panel of B denotes the representative Western blots. Data are mean ± s.e.m. Statistical analysis was performed with unpaired (A) or paired (B) Student’s t-test. *P < 0.05.
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   Figure 2

   Immunofluorescent staining of cultured amnion cells with antibodies against vimentin (green; A, C, D, F), cytokeratin (red; E and F) and CD45 (red; B and C). The nuclei were stained with DAPI (blue).
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   Figure 3

   Effects of cortisol on COL3A1 mRNA and protein abundance in human amnion fibroblasts. (A) Effects of cortisol (0, 0.01, 0.1 and 1 μM, 24 h) on COL3A1 mRNA (n = 6) and protein (n = 6) abundance. (B) Time course effects of cortisol (1 μM; 0, 3, 6, 12 and 24 h) on COL3A1 mRNA (n = 5) and protein (n = 5) abundance. (C and D) Effect of mRNA transcription inhibitor DRB (1 µM, n = 4) or protein translation inhibitor CHX (1 µM, n = 5) on cortisol (1 μM; 24 h)-induced decreases in COL3A1 protein abundance. The upper panels of A, B, C and D denote representative Western blots. Data are mean ± s.e.m. Statistical analysis was performed with one-way ANOVA test. *P < 0.05, **P < 0.01, ***P < 0.001.
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   Figure 4

   Role of GR and 11β-HSD1 in glucocorticoid-induced decreases in COL3A1 protein abundance in human amnion fibroblasts. (A) GR antagonist RU486 (1 μM) blocked cortisol (1 μM, 24 h)-induced decreases in COL3A1 protein abundance n = 5. (B) siRNA-mediated knock-down of GR attenuated cortisol (1 μM, 24 h)-induced decreases in COL3A1 protein abundance n = 5. (C) 11β-HSD1 inhibitor 10j (1 µM) blocked cortisone (5 µM, 24 h)-induced decreases in COL3A1 protein abundance n = 5. (D) siRNA-mediated knock-down of 11β-HSD1 attenuated cortisone (5 µM, 24 h)-induced decreases in COL3A1 protein abundance n = 4. The upper panels of A, B, C and D denote representative Western blots. Data are mean ± s.e.m. Statistical analysis was performed with one-way ANOVA test. **P < 0.01.
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   Figure 5

   Effects of proteasome and lysosome pathway inhibitors on cortisol-induced decreases in COL3A1 protein abundance in human amnion fibroblasts. (A) Proteasome pathway inhibitor MG132 (10 nM) blocked cortisol (1 µM, 24 h)-induced reduction in COL3A1 but not COL1A1 protein abundance, whereas the lysosome pathway inhibitor CQ (50 µM) blocked cortisol (1 µM, 24 h)-induced reduction in COL1A1 but not COL3A1 protein abundance n = 5. (B) siRNA-mediated knock-down of ATG7 blocked cortisol (1 µM)-induced reduction in COL1A1 but not COL3A1 protein abundance n = 4. (C) Bortezomib (50 nM) blocked cortisol (1 µM, 24 h)-induced reduction in COL3A1 protein abundance (n = 5). (D) Ubiquitin-activating Enzyme E1 inhibitor PYR-41 (5 µM) blocked cortisol (1 µM, 24 h)-induced reduction in COL3A1 protein abundance n = 5. Upper panels of A, B, C and D denote representative Western blots. Data are mean ± s.e.m. Statistical analysis was performed with one-way ANOVA test. *P < 0.05, **P < 0.01.
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   Figure 6

   COL3A1 ubiquitination by cortisol in human amnion fibroblasts. Cortisol (1 µM, 24 h) increased the ubiquitination of COL3A1 in the presence of MG132 in human amnion fibroblasts as revealed by Western blotting n = 3. The upper panel denotes representative Western blots. Data are mean ± s.e.m. Statistical analysis was performed with paired Student’s t-test. *P < 0.05.

• © 2018 Society for Endocrinology